Liquid MALDI MS Analysis of Complex Peptide and Proteome

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Liquid MALDI MS analysis of complex peptide and proteome samples Kanjana Wiangnon, and Rainer Cramer J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00148 • Publication Date (Web): 15 Jul 2016 Downloaded from http://pubs.acs.org on July 16, 2016

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Journal of Proteome Research

Liquid MALDI MS analysis of complex peptide and proteome samples Kanjana Wiangnon and Rainer Cramer* Department of Chemistry, University of Reading, Whiteknights, Reading, RG6 6AD, UK

*Corresponding author: Prof. Rainer Cramer Department of Chemistry University of Reading Whiteknights Reading RG6 6AD UK Phone: 0044-118-378-4550 Email: [email protected]

Keywords: liquid support matrix, liquid MALDI, MALDI-TOF, QTOF, mass spectrometry, LCMALDI, automation, MALDI-PSD, MALDI-CID, MALDI MS/MS

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Abstract Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is well-known to be a powerful technique for the analysis of biological samples. By using glycerol-based liquid support matrices (LSMs) instead of conventional MALDI matrices the power of this technique can be extended further. In this study, we exploited LSMs for the identification of complex samples, i.e. the Lactobacillus proteome and a bovine serum albumin (BSA) digest. Liquid and solid MALDI samples were manually and robotically prepared by coupling a nanoflow high performance liquid chromatography (nanoHPLC) system to an automated MALDI sample spotting device. MS and MS/MS data were successfully acquired at the femtomole level using TOF/TOF as well as Q-TOF instrumentation and used for protein identification searching sequence databases. For the BSA digest analysis, liquid MALDI samples resulted in peptide mass fingerprints which led to a higher confidence in protein identification compared to solid (crystalline) MALDI samples. However, post-source decay (PSD) MS/MS analysis of both the proteome of Lactobacillus plantarum WCFS1 cells and BSA digest showed that further optimization of the formation and detection of peptide fragment ions is still needed for liquid MALDI samples as the MS/MS ion search score was lower than for the solid MALDI samples, reflecting the poorer quality of the liquid MALDI-PSD spectra, which can be attributed to the differences in PSD parameters and their optimization that is currently achievable.


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1. Introduction Mass spectrometry (MS) is a key tool in proteomic research, enabling the identification, characterization and quantification of peptides and proteins. The most common soft ionization methods in MS are matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). The benefits of MALDI as an ionization technique include high sensitivity (low femtomole range or lower) and high tolerance to contaminants such as salts, buffers and other impurities. The sample preparation is simple and requires only small amounts of analyte. In addition, there is also the potential of recovering residual amounts of analyte. The versatility of MALDI MS has been demonstrated in many applications. 1-3 In general, matrices and solvents for MALDI sample preparations are carefully chosen for optimal analyte detection. Matrices such as 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4hydroxycinnamic acid (CHCA) have been widely employed for solid (crystalline) MALDI samples. However, such solid MALDI samples are not well suited for quantitative measurements and/or automated data acquisition because of heterogeneous crystallization, resulting in highly variable ion yields and desorption/ionization characteristics. As an alternative to the traditional matrices and solid MALDI samples, liquid support matrices (LSMs) provide increased shot-toshot reproducibility at low sample consumption, leading to prolonged and stable ion signals.4-7 Liquid matrices are often based on the dissolution of an acidic solid matrix with a basic organic reagent such as 3-aminoquinoline (3-AQ) as a solubilizing assistant.4-12 The use of a (viscous) liquid with low-volatility such as glycerol in conjunction with this acid/base system supports the sample to remain liquid, even under vacuum conditions.4-7 In contrast to solid MALDI samples, liquid MALDI samples give greater homogeneity with a more constant spot morphology after



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irradiation due to the self-healing and renewing properties of the liquid samples. As the spot remains liquid under vacuum, a prolonged period of ion generation and detection is also possible in (high-)vacuum MALDI instruments such as axial TOF and Q-TOF instruments. Thus, this approach facilitates the application of MALDI MS for automation and quantification using commercial instrumentation.5-7 In MS-based studies of complex proteomes, MS is often coupled to liquid chromatography (LC) separation either in an on-line or off-line format. LC coupling to MALDI is typically carried out off-line with the MALDI matrix and LC eluent fractions being mixed and spotted onto the MALDI target. A few studies have attempted to analyze complex proteomic samples with this approach and achieved protein identification at the low-femtomole level using proteolytic digests.13-15 To date, most proteomics research has focused on using on-line LC interfaced with ESI MS. However, there are some notable advantages in interfacing LC with MALDI rather than ESI for the analysis of a complex peptidic digest mixture. With on-line LC-ESI MS, the time interval is often too short for the acquisition of fragment ion spectra of all the sample components. This time constraint does not apply to off-line LC-MALDI MS. The sample can be stored on the MALDI target plate for at least several days without analyte degradation. Another potential benefit is the detection of mainly singly charged ions, whereas ions are typically detected as multiply charged species in ESI MS. This difference to ESI MS enables easy interpretation of the mass spectra. However, due to the differences between MALDI and ESI, they each have specific advantages over the other. Therefore, combining the data from both LC-ESI MS/MS and LCMALDI MS/MS approaches have improved the number of proteins identified and the protein coverage of complex protein digest mixtures.16-18 4 ACS Paragon Plus Environment

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The aim of the presented research has been the optimization of liquid MALDI sample preparation for the identification of complex proteomic samples using bottom-up proteomics as the analytical strategy with bovine serum albumin (BSA) and Lactobacillus proteome digests as analytes. As with ESI-based proteomics the analysis of a complex protein sample involved the use of a nanoflow HPLC system for the separation of the digest peptides. The HPLC system was coupled to an automated sample spotting robot that simultaneously co-spots liquid MALDI matrix aliquots with HPLC fractions. The ability to use nanoHPLC-MALDI MS and MS/MS for the identification of a BSA digest resulted in its successful identification with high protein sequence coverage. In the initial experiments badly resolved and unidentifiable high-intensity peaks appeared in post-source decay (PSD) MS/MS spectra acquired on an axial TOF/TOF instrument. These peaks and their impact on protein identification were further investigated in this study.



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2. Experimental Section 2.1 Materials Peptide calibration standard II (cat.no. 222570) and CHCA were purchased from Bruker Daltonics (Coventry, UK). BSA, Glu-1-fibrinopeptide B (Glu-Fib), 3-aminoquinoline (3-AQ), ammonium phosphate (AP), ammonium citrate (AC), ammonium bicarbonate (ABC), glycerol, dithiothreitol (DTT), and iodoacetamide (IAA) were obtained from Sigma-Aldrich (Poole, UK). Sequencing-grade modified trypsin was purchased from Promega (Southampton, UK). Trifluoroacetic acid (TFA) was from Thermo Scientific (Rockford, IL, USA). The organic solvents used were also obtained from Sigma-Aldrich while the HPLC gradient-grade water came from Thermo Fisher Scientific (Loughborough, UK). All chemicals and solvents were used without further purification. L. plantarum WCFS 1 was originally isolated from human saliva (National Collection of Industrial and Marine Bacteria, Aberdeen, UK). It was kindly donated by Mr. Pornpoj Srisukchayakul (Food and Nutritional Sciences, University of Reading, Reading, UK). 2.2 Analyte Preparations A tryptic digest of BSA was prepared by dissolving 10 µg of BSA in 50 mM ABC. DTT was added to achieve a final concentration of 10 mM and the mixture was incubated at 45oC for 45 min to cleave disulphide bridges. Subsequently, the cleaved disulphide bridges were alkylated by reaction with 30 mM IAA in the dark for 45 min. The excess IAA was quenched by the addition of DTT for 45 min. Trypsin was then added to the sample in a ratio of 1:50 (w/w) (Trypsin:BSA) and the sample was left overnight at 37°C. The digestion was stopped by adding TFA to bring the pH of the mixture to 500 and a limit of 300 peaks selected. The WARP-LC software (version 1.1; Bruker Daltonics) was used to establish a peak list of an entire LC experiment after peak selection. WARP-LC was then used for the automatic selection of peptide signals and their subsequent MS/MS analysis. Automated MS/MS data acquisition was performed by using the LIFT technology of the Ultraflex TOF/TOF instrument. For this, LIFT spectra were obtained from the accumulation of 300 parent ion spectra and 500 single-shot fragment ion spectra for each selected parent ion. A maximum of 10 parent ion signals per fraction were analysed starting preferentially in the range m/z 800-3500. The LIFT fragment ion peaks were picked using FlexAnalysis after baseline subtraction (median with a 0.8 flatness) and smoothing (SavitzkyGolay; 4 cycles with a width of m/z 0.15). Peaks were selected up to a maximum of 200 with a 11 ACS Paragon Plus Environment


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peak S/N ratio of ≥ 3 using the centroid algorithm. MS and MS/MS spectra were sent by WARPLC to the BioTools software version 3.0 (Bruker Daltonics) as combined peak lists for database searching (see section 2.6). Q-TOF data acquisition, processing and analysis Q-TOF measurements were performed on a MALDI Q-TOF Premier mass spectrometer (Waters Corporation, Wilmslow, UK) in positive ion V mode using a laser emitting at 337 nm with a repetition rate of 20 Hz. The spectra were collected with cell entrance at 1.5 V, cell exit at -10.3 V, sample plate at 0 V, extraction at 9.0 V, hexapole at 9.9 V, aperture 0 at 3.3 V, and ion guide at 1.5 V. The detector was operated at a voltage of 1800 V. Q-TOF MS data acquisition resulted from data accumulation of MS scans over 1 min at a scan rate of 1 scan per second. For the solid matrix, the MALDI mass spectra were acquired with the laser irradiation moving in a spiral pattern over the MALDI sample using a step rate of 2 Hz and a step width of 3.75 µm. For the liquid matrix, the laser beam was fixed in the centre of the droplet without any movement. The collision energy voltage was set to 4.9 V and laser pulse energies were optimized to give the highest signal intensity. MassLynx version 4.1 software was used to process the mass spectra. A spectrum was created by summing up the intensities of the total ion chromatograms (TIC) over one minute. The baseline was subtracted and the S/N ratio was calculated. For MS/MS data acquisition, collision-induced dissociation (CID) was performed with the collision energy voltage settings in the range of 65-80 V. CID fragment ions were collected with a scan rate of 1 s and an inter-scan delay of 0.02 s. The mass window for transmission of precursor ions was set to 4.7 for low mass resolution and 15.0 for high mass resolution. The mass 12 ACS Paragon Plus Environment

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spectra were processed using the Savitzky-Golay smoothing method for a 3 channel window width with 2 iterations while baseline subtraction was done with a polynomial order of 15 and a tolerance of 0.01. 2.6 Database Searching For BSA analysis, MS and MS/MS peak lists were automatically searched against the Swiss-Prot protein database (version 57.60) using Mascot Server 2.4.1 (Matrix Science, London, UK). For L. plantarum, the MS/MS data was searched against the in-house database of Lactobacillus plantarum WCFS1, containing 3088 protein entries (934,303 residues), which was downloaded on March 31, 2014 from the HAMAP (High-quality Automated and Manual Annotation of microbial

Proteomes)

database

repository

website

(http://hamap.expasy.org/proteomes/LACPL.html). Peptide mass fingerprinting (PMF) was performed with a mass tolerance of 100 ppm, allowing for up to 1 missed cleavage, with a fixed modification of cysteine carbamidomethylation and variable modifications of acetylation at the N-terminus and methionine oxidation. MS/MS ions searching was performed with the peptide mass tolerance set to 100 ppm and the MS/MS ion tolerance to 0.7 Da. The modifications of fixed carbamidomethylation of cysteines and variable oxidation of methionines were selected. A maximum of 1 missed cleavage was allowed.



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3. Results and Discussion Glycerol-based liquid support matrices (LSMs) have been successfully used as MALDI matrices for peptide and protein analysis, with the benefit of high reproducibility due to the greater homogeneity and prolonged ion signal. This advantage facilitates the identification of proteins by peptide mass mapping using automated sample preparation and data acquisition.6 A recent study even used (glycerol-based) LSMs for producing multiply charged peptide and protein ions using an AP ion source.19 This study describes a novel approach using liquid MALDI for the analysis of complex protein digests with the automated step of nanoHPLC and MALDI sample spotting being off-line from the MALDI-TOF MS(/MS) analysis. Several LC-MALDI MS approaches have been proposed using solid MALDI samples but there are no reports where liquid (LSM) matrices have been used with LC-MALDI. Zenobi and co-workers have however demonstrated the use of an ionic liquid as the matrix for automated co-deposition with the analyte, using a self-built device, though not in the context of coupling LC and MALDI MS, e.g. for the analysis of complex peptide mixtures.15 For this work, the performance of both liquid and solid MALDI sample preparations were tested using the workflow described. While the preparation of the liquid matrix solution requires the addition of a couple of more components (glycerol and base), these are not costly and the additional time needed for the preparation of the liquid matrix stock solution is negligible. As homogeneity is a key aspect of liquid MALDI, sample spots were viewed under a microscope after deposition by the automated spotting device. It was observed that these spots were still in the liquid form even when placed under vacuum in the mass spectrometer, whereas equivalent 14 ACS Paragon Plus Environment

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spots made using the solid matrix CHCA appeared as fully dried small crystals after evaporation of the volatile solvent (Figure 1). Mascot search results of the peptide mass fingerprints using these two MALDI sample preparations are compared in Table 1. The total number of selected peaks were 153+66 and 172+74 for the liquid and solid MALDI samples, respectively. For the liquid MALDI analysis, the average sequence coverage and Mascot score for the BSA digest were 65+3% and 258+11, respectively, while the same experiment conducted with the solid MALDI sample gave 60+3% and 207+20, respectively. One of the keys to successful PMF with high scores is the prevention of unwanted clusters and adducts of the matrix compounds as discussed in a previous report.6 The lower number of selected peaks obtained using the liquid sample preparation is in agreement with the observation that this liquid MALDI sample preparation can reduce salt adduct and matrix cluster ion signals.6, 20 Some of these beneficial effects can be attributed to the presence of ammonium phosphate in the liquid MALDI sample. 21-26

Similarly, the addition of optimal amounts of basic organic compounds to the matrix acid

has the effect of decreasing chemical noise, alkali adducts and matrix clusters, further increasing sensitivity. 5, 6, 27, 28 In Table 1, intensity coverage, a parameter calculated by the software Biotools, represents the sum of the intensities of the matched peaks divided by the sum of the intensities of the peaks that have been selected (multiplied by 100). For the liquid MALDI samples, intensity coverage was 52+7%, compared to 42+5% for the solid MALDI samples. Some of this difference in intensity coverage could be due to the disparity in homogeneity of the samples. For the solid samples, laser shots were taken at random positions. The non-homogeneous distribution of analyte within the crystals easily leads to a reduction in the overall S/N as many laser shots typically result in spectra with little or no analyte ion signal, even if the operator (or automated acquisition) is well 15 ACS Paragon Plus Environment


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experienced in searching analyte-containing ‘sweet spots’. In contrast there is no requirement to search for a ’sweet spot’ with the liquid samples due to their homogenous and self-healing properties. Therefore, higher peptide ion signal can be obtained from a single laser position with a persistent analyte ion signal that fluctuates very little from shot to shot. For MS/MS analysis of a BSA digest, Table 2 presents the database search results for both MALDI sample preparations. The number of matched peptides was the same (25+5) for both sample preparations, while the protein sequence coverage was higher for the liquid MALDI samples (50+8%) than for the solid MALDI samples (47+9%). However, liquid MALDI gave a poorer Mascot score (965+320) than solid MALDI (1299+244). In general, a higher peptide match score was obtained when using solid MALDI (i.e. for 15 out of the 20 peptide ions that were matched in both cases). These results reflect the poorer b- and y-ion assignment in liquid MALDI. A lower mass accuracy is partially the reason for obtaining lower Mascot scores in liquid MALDI. For example, the solid MALDI PSD MS/MS spectrum of the peptide ion at m/z 1907.9 led to a BSA peptide match, whereas the respective liquid MALDI spectrum could not be successfully matched because the measured fragment ion masses were outside the mass tolerance window of ±0.7 Da, although fragment ion signal intensities were greater (Figure 2). One possible explanation for this low fragment ion mass accuracy lies with the calibration of the LIFT. The LIFT is normally calibrated during the annual preventative maintenance visit by the service engineer using crystalline MALDI samples only and is therefore most likely inadequate for liquid MALDI with its generally thicker sample and different instrument parameter settings and calibration. Some of the BSA peptides identified using liquid MALDI, for example, m/z 927.4, showed more intense ion signals and achieved a higher ion score with PSD than using solid MALDI (Figure 16 ACS Paragon Plus Environment

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3). A comparison was made of all the peptides observed using three replicate analyses with each matrix, but only where the peptide ion was found in more than one replicate were the files selected and data combined. This comparison revealed that seven ions below m/z 1200 were detected when using liquid MALDI as opposed to only three peptide ions found for solid MALDI (see Table 3). There were 20 peptides present in both data sets. Five peptides were exclusively found in solid MALDI, while eight peptides were found only in liquid MALDI (Table 3). The peptides exclusively found using liquid MALDI were all detected below m/z 1600 while all apart from one of the peptide ions exclusively obtained with solid MALDI were in the range above this m/z value. These results demonstrate the effectiveness of liquid MALDI in the detection of peptide ions in the low mass range, as has been shown previously.6 Again, a possible reason for this is the reduction in matrix interference seen in the low mass region. Next, a similar approach was used for the analysis of the proteome of Lactobacillus cells. Here, liquid MALDI MS/MS returned a lower overall number of identifications (84 proteins) as compared to the 188 proteins identified using solid MALDI MS/MS (Table 4). The relatively low number of protein identifications can be due to the composition of the Lactobacillus database, which has a lower number of residues per entry (~300) compared to other (wellcurated) databases and has an extremely low number of only 41 entries with proteomic or transcriptomic evidence (http://hamap.expasy.org/proteomes/LACPL.html; accessed on 17th June 2016). The above finding led to an investigation into the quality of the PSD spectra using liquid MALDI. Notably, the PSD MS/MS spectra revealed a strong unresolved peak near the theoretical m/z values of the bn-1/yn-1 peptide fragment ions, as shown in Figure 4. In around half of the cases these peaks were closer to the theoretical yn-1 m/z value while for the other half they were closer to the bn-1 or bn-1+H2O values. However, unresolved peaks were not present in all 17 ACS Paragon Plus Environment


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peptide spectra. A total of 42% of the BSA spectra had these unresolved peaks while the rest did not. The intensities of those peaks also varied with each peptide. For Lactobacillus proteins, there were unresolved peaks in 25% of the total MS/MS measurements using liquid MALDI, of which 10% (i.e. 2.5 % of the total MS/MS measurements) could be matched by Mascot. Further experiments were carried out to investigate the cause of the unresolved peaks, what they were and how to prevent them from having an adverse effect on the MS/MS analysis. One early indication was that the unresolved peaks are somehow related to the presence of AP in the liquid matrix. As mentioned earlier, the ammonium salt suppresses alkali-ion adduct formation for peptides and plays a similarly important role in the protonation and deprotonation of oligonucleotides 21, 24, 29 and phosphopeptides. 25 An experiment to test this hypothesis was conducted using the peptide angiotensin I from the peptide calibration standard II. AP was either used, omitted from the liquid MALDI preparation or replaced with ammonium citrate (AC). The resulting PSD spectra are shown in Figure 5. Without any ammonium salts (Figure 5(c)) the spectra did not show a large number of unresolved peaks but a few intense PSD ion signals were observed at around m/z 1080 which do not correlate to angiotensin I fragments. With AC (Figure 5(b)), unresolved peaks were observed similar to those seen when AP was used (Figure 5(a)) but these peaks were at lower as well as higher m/z values. In contrast, the analogous experiment using the solid MALDI matrix showed no unresolved peaks in the spectrum (Figure 5(d)). Some of these spectral changes could be due to different chemistries provided by these additives or simply due to physical property changes of the liquid MALDI sample as a result of a change in surface tension or accumulation of ions on the surface. Unfortunately, no conclusive correlation to the appearance of these unwanted spectral features could be found. However, without ammonium salts in the 18 ACS Paragon Plus Environment

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liquid matrix the protein identification was adversely affected due to the increased presence of salt adducts and matrix clusters in the MS spectrum. Thus, the use of AP is still desirable in liquid MALDI. Interestingly, in the presence of ammonium salt, the laser energy threshold value needed for the generation of MALDI analyte ions was higher than without ammonium salt: Eions(AP) > Eions(AC) > Eions(no ammonium salt). An explanation for this can possibly be found in the negligible UV absorption for ammonium salts and a potential accumulation of their ionic constituents preferentially on the surface of the liquid MALDI droplet, thus significantly lowering the absorbance of the irradiated sample volume and demanding higher laser energy for successful MALDI ion generation. Previous reports suggest that ammonium cations as monovalent and polarizable ions show a high propensity to be near (aqueous) droplet surfaces, supporting the possibility of an absorbance dilution effect.

30, 31

In particular, recent data show

that water droplets doped with specific ionic compounds can even produce thin films on the droplets’ surface with a thickness of approximately 1 µm

32

, which is greater than the ablation

depth in liquid UV-MALDI (and the penetration depth of the laser light), indicating that the absorbance can be significantly reduced by the creation of such thin films if they consist of lowabsorbing compounds. Higher laser energy requirements generally result in higher internal energies of the analyte ions and therefore greater unimolecular (metastable) decay. However, beside the sample preparation, instrumental and fragmentation parameters can also affect the amount and type of detected fragment ions. To investigate this, the standard peptide angiotensin I was examined using CID on a hybrid Q-TOF mass spectrometer. Spectra were acquired from 100 fmol on target using both solid and liquid MALDI. Fragment ion lists obtained from the MS/MS spectra were compared with the theoretical fragment ion masses of 19 ACS Paragon Plus Environment


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angiotensin I calculated using Protein Prospector (http://prospector.ucsf.edu) at a fragment ion mass tolerance of less than + 0.2 Da. The CID spectra of angiotensin I acquired with liquid MALDI gave greater peptide sequence information and higher intensities of fragment ion signals (which were also of equivalent quality) compared to the spectra obtained by solid MALDI on the same instrument (Figure 6(a,b)). The LSM MS/MS spectra, however, showed a high level of background noise (Figure 6(b)), whose ions do not match any theoretical CID fragment ions and could originate from chemical background noise from the matrix as more laser power was required with the liquid than solid sample. It is interesting to note that no unresolved intense peaks were observed in the liquid MALDI CID spectra using the Q-TOF instrument (Figure 6(b)). Unresolved intense fragment ion peaks appeared only in PSD spectra acquired on the Ultraflex axial TOF instrument (Figure 6(d)). In the Ultraflex instrument, ions normally fragment before entering the LIFT cell where they then gain kinetic energy from the LIFT acceleration, resulting in kinetic energies in the range of 19-27 keV. In the presence of an ammonium salt, fragmentation could be delayed and occur late when the ions are in the LIFT cell or after they have left it. Here, the fragment ions may have a kinetic energy below 19 keV. However, if the ions were to fragment late, they would acquire lower kinetic energies and travel much slower through the (reflector) TOF resulting in a shift to an apparently higher mass and with poorer resolution.

4. Conclusions This study investigated the use of liquid MALDI with an automated nanoHPLC MALDI MS(/MS) set-up. The set-up was successfully used for the analysis of trypsin digests of BSA and Lactobacillus proteins. For the analysis of a BSA digest, the liquid MALDI sample preparation yielded PMFs with a higher sequence coverage and a higher Mascot score than the equivalent 20 ACS Paragon Plus Environment

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solid MALDI sample preparation, while the relative performances in MS/MS ion searches were similar. For the Lactobacillus proteome, liquid MALDI samples can identify highly abundant proteins. However, when using liquid MALDI, PSD spectra show intense unresolved peaks around the yn-1/bn-1 fragment ions, particularly in the presence of ammonium salts. These peaks were not seen when spectra were acquired on a Q-TOF instrument using CID. Thus, future work will center on the optimization of the analysis of complex proteomic samples using liquid MALDI and automated LC-MALDI MS/MS on a Q-TOF instrument, and further investigate the origin of the intense unresolved PSD fragment ion signals observable with liquid MALDI.

Acknowledgements This research was supported by the Royal Thai Government. We would like to acknowledge the Chemical Analysis Facility at the University of Reading for access to the Ultraflex TOF/TOF instrument.

Conflict of Interest Disclosure The authors declare no competing financial interest.



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References 1

R. Cramer, K. Dreisewerd. in The Encyclopedia of Mass Spectrometry (Eds.: M. L. Gross, R. M. Caprioli), Elsevier, Amsterdam, The Netherlands 2007, pp. 646.

2

D. E. Hoffmann, V. Stroobant. Mass Spectrometry: principles and applications, 3rd ed., John Wiley & Sons, Inc, Chichester, England, 2007.

3

D. S. Nagra, L. Li. Liquid chromatography-time-of-flight mass spectrometry with continuous-flow matrix-assisted laser desorption ionization. J Chromatogr A 1995, 711, 235.

4

E. T. P. Sze, T. W. D. Chan, G. Wang. Formulation of Matrix Solutions for Use in Matrix-Assisted Laser Desorption/Ionization of Biomolecules. J Am Soc Mass Spectrom 1998, 9, 166.

5

M. Towers, R. Cramer. Liquid Matrices for Analyses by UV-MALDI Mass Spectrometry. Spectroscopy 2007, 22, 24.

6

R. Cramer, S. Corless. Liquid ultraviolet matrix-assisted laser desorption/ionization-mass spectrometry for automated proteomic analysis. Proteomics 2005, 5, 360.

7

M. Palmblad, R. Cramer. Liquid Matrix Deposition on Conductive Hydrophobic Surfaces for Tuning and Quantitation in UV-MALDI Mass Spectrometry. J Am Soc Mass Spectrom 2007, 18, 693.

8

Y. Fukuyama, K. Takeyama, S. Kawabata, S. Iwamoto, K. Tanaka. An optimized matrix-assisted laser desorption/ionization sample preparation using a liquid matrix, 3-aminoquinoline/alpha-cyano-4-hydroxycinnamic acid, for phosphopeptides. Rapid Commun Mass Spectrom 2012, 26, 2454.

9

K. Kaneshiro, Y. Fukuyama, S. Iwamoto, S. Sekiya, K. Tanaka. Highly Sensitive MALDI Analyses of Glycans by a New Aminoquinoline-Labeling Method Using 3Aminoquinoline/α-Cyano-4-hydroxycinnamic Acid Liquid Matrix. Anal Chem 2011, 83, 3663.

10

S. Sekiya, K. Taniguchi, K. Tanaka. On-target separation of analyte with 3aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix for matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 2012, 26, 693.

11

T. Shuo, N. Koshikawa, D. Hoshino, T. Minegishi, H. Ao-Kondo, M. Oyama, S. Sekiya, S. Iwamoto, K. Tanaka, M. Seiki. Detection of the Heterogeneous OGlycosylation Profile of MT1-MMP Expressed in Cancer Cells by a Simple MALDI-MS Method. PLoS ONE 2012, 7, e43751. 22 ACS Paragon Plus Environment

Page 23 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


12

V. S. K. Kolli, R. Orlando. A new matrix for matrix-assisted laser desorption/ionization on magnetic sector instruments with point detectors. Rapid Commun Mass Spectrom 1996, 10, 923.

13

E. Mirgorodskaya, C. Braeuer, P. Fucini, H. Lehrach, J. Gobom. Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: sample preparation, data analysis, and application to the analysis of complex peptide mixtures. Proteomics 2005, 5, 399.

14

F. Pereira, X. Niu, A. J. deMello. A Nano LC-MALDI Mass Spectrometry Droplet Interface for the Analysis of Complex Protein Samples. PLoS ONE 2013, 8, e63087.

15

S. Weidmann, S. Kemmerling, S. Mädler, H. Stahlberg, T. Braun, R. Zenobi. Ionic liquids as matrices in microfluidic sample deposition for high-mass matrixassisted laser desorption/ionization mass spectrometry. Eur J Mass Spectrom 2012, 18, 279.

16

W. M. Bodnar, R. K. Blackburn, J. M. Krise, M. A. Moseley. Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage. J Am Soc Mass Spectrom 2003, 14, 971.

17

Y. Yang, S. Zhang, K. Howe, D. B. Wilson, F. Moser, D. Irwin, T. W. Thannhauser. A Comparison of nLC-ESI-MS/MS and nLC-MALDI-MS/MS for GeLC-Based Protein Identification and iTRAQ-Based Shotgun Quantitative Proteomics. J Biomol Tech 2007, 18, 226.

18

S. J. Hattan, J. Marchese, N. Khainovski, S. Martin, P. Juhasz. Comparative Study of [Three] LC−MALDI Workflows for the Analysis of Complex Proteomic Samples. J Proteome Res 2005, 4, 1931.

19

R. Cramer, A. Pirkl, F. Hillenkamp, K. Dreisewerd. Liquid AP-UV-MALDI Enables Stable Ion Yields of Multiply Charged Peptide and Protein Ions for Sensitive Analysis by Mass Spectrometry. Angew Chem Int Ed 2013, 52, 2364.

20

R. Cramer, J. Gobom, E. Nordhoff. High-throughput proteomics using matrixassisted laser desorption/ ionization mass spectrometry. Expert Rev Proteomics 2005, 2, 407.

21

Y. F. Zhu, N. I. Taranenko, S. L. Allman, S. A. Martin, L. Haff, C. H. Chen. The Effect of Ammonium Salt and Matrix in the Detection of DNA by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry. Rapid Commun Mass Spectrom 1996, 10, 1591.

22

I. P. Smirnov, X. Zhu, T. Taylor, Y. Huang, P. Ross, I. A. Papayanopoulos, S. A. Martin, D. J. Pappin. Suppression of α-Cyano-4-hydroxycinnamic Acid Matrix Clusters and Reduction of Chemical Noise in MALDI-TOF Mass Spectrometry. Anal Chem 2004, 76, 2958. 23 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 36

23

X. Zhu, I. A. Papayannopoulos. Improvement in the Detection of Low Concentration Protein Digests on a MALDI TOF/TOF Workstation by Reducing αCyano-4-hydroxycinnamic Acid Adduct Ions. J Biomol Tech 2003, 14, 298.

24

G. J. Currie, J. R. Yates. Analysis of oligodeoxynucleotides by negative-ion matrix-assisted laser desorption mass spectrometry. J Am Soc Mass Spectrom 1993, 4, 955.

25

J. M. Asara, J. Allison. Enhanced detection of phosphopeptides in matrixassisted laser desorption/ionization mass spectrometry using ammonium salts. J Am Soc Mass Spectrom 1999, 10, 35.

26

Y. C. L. Li, S.-w. Cheng, T. W. D. Chan. Evaluation of ammonium salts as comatrices for matrix-assisted laser desorption/ionization mass spectrometry of oligonucleotides. Rapid Commun Mass Spectrom 1998, 12, 993.

27

M. Zabet-Moghaddam, E. Heinzle, M. Lasaosa, A. Tholey. Pyridinium-based ionic liquid matrices can improve the identification of proteins by peptide massfingerprint analysis with matrix-assisted laser desorption/ionization mass spectrometry. Anal Bioanal Chem 2006, 384, 215.

28

C. D. Calvano, S. Carulli, F. Palmisano. Aniline/alpha-cyano-4-hydroxycinnamic acid is a highly versatile ionic liquid for matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 2009, 23, 1659.

29

U. Pieles, W. Zürcher, M. Schär, H. E. Moser. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a powerful tool for the mass and sequence analysis of natural and modified oligonucleotides. Nucleic Acids Res 1993, 21, 3191.

30

S. Gopalakrishnan, P. Jungwirth, D. J. Tobias, H. C. Allen. Air−Liquid Interfaces of Aqueous Solutions Containing Ammonium and Sulfate: Spectroscopic and Molecular Dynamics Studies. J Phys Chem B 2005, 109, 8861.

31

C. Tian, S. J. Byrnes, H.-L. Han, Y. R. Shen. Surface Propensities of Atmospherically Relevant Ions in Salt Solutions Revealed by Phase-Sensitive Sum Frequency Vibrational Spectroscopy. J Phys Chem Lett 2011, 2, 1946.

32

X. Zeng, Y. Zhang, Z. Xia, L. Wang, C. Wang, Y. Huang, R. Shen, W. Wen. Surface evolution of manganese chloride aqueous droplets resulting in selfsuppressed evaporation. Sci Rep 2015, 5.


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Tables Table 1. Comparison of peptide mass fingerprints of a BSA digest using solid and liquid MALDI sample preparations (n=3, searching the Swiss-Prot database).

Picked peaks*

Matched peaks

Sequence coverage (%)

Intensity coverage ** (%)

Mascot score***

Liquid MALDI

153 + 66

46 + 9

65 + 3

52 + 7

258 + 11

Solid MALDI

172 + 74

44 + 5

60 + 1

42 + 5

207 + 20

* Peaks were picked above m/z 800 with a signal-to-noise ratio of ≥ 6. ** This parameter is calculated by Biotools and represents the sum of the intensities of the matched peaks divided by the sum of the intensities of the peaks selected (multiplied by 100). *** Mascot scores greater than 61 are significant.



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Table 2. Comparison of MS/MS ion searches of a BSA digest using solid and liquid MALDI sample preparations (n=3, searching the Swiss-Prot database; peaks were selected if the signalto-noise ratio was ≥ 3).

Number of acquired MS/MS spectra

Matched peptides

Protein sequence coverage (%)

Mascot score

Liquid MALDI

180 + 49

25 + 5

50 + 8

965 + 320

Solid MALDI

255 + 73

25 + 5

47 + 9

1299 + 244


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Table 3. List of BSA peptide ions found in more than one of three replicates for solid and liquid MALDI sample preparations.

Peptide ions (m/z) 922.53 927.52 1014.62 1107.48 1138.51 1142.81 1163.69 1249.65 1283.60 1305.72 1399.69 1415.70 1419.76 1439.87 1443.65 1478.50 1479.76 1502.64 1511.74 1532.80 1554.68 1567.81 1576.72 1639.89 1673.79 1724.84 1740.81 1747.60 1749.66 1880.71 1888.86 1907.88 2458.30

Occurrences in solid MALDI

Peptide sequence IETMREK YLYEIAR QTALVELLK EAC(Carbamidomethyl)FAVEGPK C(Carbamidomethyl)C(Carbamidomethyl)TESLVNR KQTALVELLK LVNELTEFAK FKDLGEEHFK HPEYAVSVLLR HLVDEPQNLIK TVMENFVAFVDK TVM(Oxidation)ENFVAFVDK SLHTLFGDELC(Carbamidomethyl)K RHPEYAVSVLLR YIC(Carbamidomethyl)DNQDTISSK ETYGDMADC(Carbamidomethyl)C(Carbamidomethyl)EK LGEYGFQNALIVR EYEATLEEC(Carbamidomethyl)C(Carbamidomethyl)AK VPQVSTPTLVEVSR LKEC(Carbamidomethyl)C(Carbamidomethyl)DKPLLEK DDPHAC(Carbamidomethyl)YSTVFDK DAFLGSFLYEYSR LKPDPNTLC(Carbamidomethyl)DEFK KVPQVSTPTLVEVSR QEPERNEC(Carbamidomethyl)FLSHK MPC(Carbamidomethyl)TEDYLSLILNR M(Oxidation)PC(Carbamidomethyl)TEDYLSLILNR YNGVFQEC(Carbamidomethyl)C(Carbamidomethyl)QAEDK EC(Carbamidomethyl)C(Carbamidomethyl)HGDLLEC (Carbamidomethyl)ADDR RPC(Carbamidomethyl)FSALTPDETYVPK HPYFYAPELLYYANK LFTFHADIC(Carbamidomethyl)TLPDTEK DAIPENLPPLTADFAEDKDVC(Carbamidomethyl)K


X

X X X X X X X X X

X X X X X X X X X X

Occurrences in liquid MALDI X X X X X X X X X X X X X X X X X X X X X X X X X X

X

X

X X X X

X


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Table 4. Comparison of MS/MS ion searches for the L. plantarum proteome analysis using solid and liquid MALDI sample preparations (n=3, searching the in house lactobacillus database).

No. of acquired MS/MS spectra

No. of matching peptides

No. of identified proteins

Liquid MALDI

2,599 + 1,219

319 + 8

84 + 4

Solid MALDI

2,785 + 1,219

888+ 139


188 + 21

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Figure Captions Figure 1. Images of a solid (A) and liquid (B) MALDI sample of a BSA digest. Both samples were robotically deposited on an AnchorChip target. Figure 2. MALDI-PSD-TOF/TOF MS/MS spectra of the tryptic BSA peptide at m/z 1907.9 using solid (a) and liquid (b) MALDI sample preparation. (c) Mass list of the fragment ions with their mass accuracy for both sample preparations. Figure 3. MALDI-PSD-TOF/TOF MS/MS spectra of the tryptic BSA peptide at m/z 927.4 using liquid (a) and solid (b) MALDI sample preparation. Figure 4. Liquid MALDI-PSD-TOF/TOF MS/MS spectra of a tryptic BSA peptide at m/z 1107.5 with a successful database match (a) and m/z 1034.6 with an unsuccessful database match (b). Both spectra show a strong unresolved peak (see also inset). Figure 5. MALDI-PSD-TOF/TOF MS/MS spectra of angiotensin I (DRVYIHPFHL) using liquid MALDI sample preparations with (a) 10 mM AP (b) 10 mM AC and (c) without AP or AC, and (d) using CHCA-based solid MALDI sample preparation. Asterisks (*) mark ion signals matching angiotensin I fragment ions. Figure 6. Fragment ion mass spectra of 100 fmol angiotensin I. Q-TOF-CID MS/MS spectra using solid (a) and liquid (b) MALDI. Axial TOF-PSD MS/MS spectra using solid (c) and liquid (d) MALDI. Spectra are annotated according to the theoretical angiotensin I fragment ion pattern.



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For TOC only


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Figure 1 144x64mm (300 x 300 DPI)

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Figure 2 76x104mm (600 x 600 DPI)


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Figure 3 41x45mm (600 x 600 DPI)



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Liquid MALDI MS Analysis of Complex Peptide and Proteome

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